Lung cancer is the leading cause of cancer-related mortality in the world. The five-year survival rate is less than 15% for patients with advanced non-small cell lung cancer (NSCLC). Lack of tumor specificity remains a major problem for chemotherapies in which side effects prevent the delivery of drug dosages needed to eliminate the majority of cancer cells. Ligand-mediated target therapy might, however, render chemotherapy more tumor specific and less toxic, and may useful for the development of novel therapies for cancer.
Even though the pharmaceutical industry has been successful in discovering many new cytotoxic drugs that are potential candidates for the treatment of cancer, this life-threatening disease still causes more than 7 million deaths every year worldwide and the number is growing (Mantyh, 2006). The clinical use of most conventional chemotherapeutics is often limited by inadequate delivery of therapeutic drug concentrations to the tumor target tissue or by severe and harmful toxic effects on normal organs. It is therefore of importance to develop novel microcarrier technologies that can be used for targeted drug delivery to tumors and thereby improve the therapeutic index of the carried drugs.
Phage display, a selection technique in which a peptide or protein is expressed as a fusion with a coat protein of bacteriophage, results in display of the fusion peptide or protein on the surface of the virion. Phage-displayed random peptide libraries provide opportunities to map B-cell epitopes (D'Mello et al., 1997; Fu et al., 1997; Scott and Smith, 1990; Wu et al., 2001; Wu et al., 2003) and protein-protein contacts (Atwell et al., 1997; Bottger et al., 1996; Nord et al., 1997; Smith et al., 1999), select bioactive peptides bound to receptors (Koivunen et al., 1999; Li et al., 1995; Wrighton et al., 1996) or proteins (Bottger et al., 1996; Castano et al., 1995; DeLeo et al., 1995; Kraft et al., 1999; Pasqualini et al., 1995), search for disease-specific antigen mimics (Folgori et al., 1994; Liu et al., 2004; Prezzi et al., 1996), and determine cell- (Barry et al., 1996; Lee et al., 2004; Mazzucchelli et al., 1999) and organ-specific peptides (Arap et al., 1998; Essler and Ruoslahti, 2002; Pasqualini et al., 1995; Pasqualini and Ruoslahti, 1996).
Liposomes were suggested as drug carriers in cancer chemotherapy (Gregoriadis et al., 1974). Since then, the interest in liposomes has increased and liposome systems are now being extensively studied as drug carriers. Three basic requirements are desired for liposomes to be used in delivering drugs specifically to cancerous tissue: (i) prolonged blood circulation, (ii) sufficient tumor accumulation, (iii) controlled drug release and uptake by tumor cells with a release profile matching the pharmacodynamics of the drug.
Initially, the research in liposome drug delivery systems suffered from very fast blood clearance by the reticuloendothelial system (RES). It was recognized that particle size, surface charge (Weinstein, 1984), and liposome composition had a strong influence on the clearance profile (e.g., incorporation of phosphatidylinositols or monosialogangliosides prolongs liposome circulation in the blood) (Allen and Chonn, 1987; Gabizon and Papahadjopoulos, 1988; Senior, 1987). This uptake may be evaded by ‘stealth’ liposomes, which preferentially exit the circulation via leaky capillaries and are predicted to accumulate in tumors exhibiting extensive neo-vascularization leading to higher concentrations and enhanced antitumor activity (Wu et al., 1993). However, liposomes were only fully recognized as successful drug delivery candidates when it was discovered that liposomes coated with the synthetic polymer polyethyleneglycol (PEG) had significantly increased half-life in the blood (Allen et al., 1991; Blume and Cevc, 1990; Klibanov et al., 1990; Papahadjopoulos et al., 1991; Senior et al., 1991). The pegylated liposomes are long circulating due to a highly hydrated and protected liposome surface, which inhibits protein adsorption and opsonization of the liposomes (Woodle and Lasic, 1992). Having solved the problems of fast opsonization and clearance, providing liposomes with up to 72 h half-life in the blood (Drummond et al., 1999), the next challenge was to get the liposomes to accumulate in the tumor tissue through active targeting. The use of targeting liposomes may potentially lead to significantly enhanced drug release at the tumor target site and increased therapeutic efficacy (Lee et al., 2004; Park et al., 2002).
The drug delivery research field has successfully constructed long circulating liposomes that accumulate in tumor tissue where the entrapped drugs then have to leak out of the liposomes by passive diffusion, unless there is an active trigger present. The use of site-specific triggers that can release drugs specifically in diseased tissue is one way of increasing drug bioavailability at the tumor target site. Another way of optimizing drug bioavailability is to obtain a higher degree of liposome accumulation by active targeting. Furthermore, the combination of active targeting with active triggering may potentially lead to significantly enhanced and specific drug release at the tumor target site (Lee et al., 2004; Park et al., 2002).
One major limitation of using monoclonal antibodies to target cancer is that the antibody molecule is relatively large with a molecular weight (MW) of 150,000. This molecule has difficulty reaching the interior of large tumor masses where the blood supply is inadequate (Dvorak et al., 1991; Jain, 1997; Shockley et al., 1991). To overcome this problem, some researchers are now developing antitumor single chain Fv (scFv) antibodies that are smaller in size, MW 25,000 (Adams and Schier, 1999; Hall et al., 1998; Wong et al., 2001). Another major problem with monoclonal antibody therapy is the nonspecific uptake of the antibody molecules into the reticuloendothelial system such as the liver, spleen, and bone marrow. The dose-limiting toxicities of radiolabeled or toxin conjugated antibody are liver and bone marrow toxicities (Neumeister et al., 2001; Thomas et al., 1995). In contrast, peptides can be considerably smaller than monoclonal antibodies and they generally do not bind to the reticuloendothelial system (Aina et al., 2005). They are chemically stable and relatively easy to manufacture. In addition, the effective tissue penetration of short synthetic peptides, in combination with their selective binding and internalizing capacity by cancer cells, make these agents ideal candidates for delivery of therapeutics such as cytotoxic drugs, oligonucleotides, toxins, and radioactive molecules. In contrast to viral delivery vectors and monoclonal antibodies, peptides are nearly invisible to the immune system and are expected to cause minimal or no side effects (Levy and Hyman, 2005). Therefore, identification of targeting ligands and development of ligand-targeted liposomes is highly desirable.
We believe that promising novel therapy for NSCLC will require tumor targeted approaches that will allow greater tumor specificity and less toxicity. Here, we describe the isolation and identification of peptides, including SP5-2 which could bind specifically to several NSCLC cell lines and human biopsy specimens from lung cancer patients. When coupled to liposomes containing vinorelbine or doxorubicin, the targeting peptide SP5-2 enhanced the therapeutic index of the drugs against human lung cancer xenografts in SCID mice. Our results indicate that this targeting peptide, and the other peptides identified in our study, have strong clinical potential as a drug delivery agents in the treatment of lung cancer.
Screening phage display libraries against specific target tissues would therefore be a direct and fast method in identifying peptide sequences, which are used for targeting of drugs, gene delivery vectors or other therapeutic agents.